In order to maintain their shape and integrity, it is critical that all types of cells contain a structural scaffold. This structure is known as the cytoskeleton and is composed of a framework of interlocking proteins. The major protein component of the cytoskeleton is actin, and the assembly of actin monomers into the cytoskeleton is highly regulated.
Many cellular processes are mediated by the cytoskeleton, especially those involving the interaction of the cell with the surrounding environment. These include cell adhesion, cell motility and cell polarity. For example, changes occurring during cell cycle progression such as those associated with surface adhesion signals are dependent on the appropriate assembly and disassembly of the cytoskeleton. Therefore, it is currently believed that the survival of the cell depends on the controlled regulation of the cytoskeleton.
RhoC, a member of the Rho subfamily of small GTPases, is a protein that has been shown to be involved in a diverse set of signaling pathways including the ultimate regulation of the dynamic organization of the cytoskeleton. The first known biological function of Rho proteins, described in Swiss 3T3 fibroblasts, was the formation of stress fibers (actin filament bundles) and focal adhesion complexes upon the addition of extracellular ligands (Ridley and Hall, Cell, 1992, 70, 389-399). These structures allow the cell to attach and pull along an extracellular substrate altering the cell's shape and position. Since then, the assembly of the cytoskeleton through the activation of Rho proteins has been demonstrated in epithelial cells, endothelial cells, astrocytes, lymphocytes, preadipocytes, platelets and neurons. While activation of cytoskeletal assembly most often results in the growth or extension of a cell, in neurons, Rho family proteins have been shown to induce neurite retraction and cause cell rounding, (Hall, Science, 1998, 279, 509-514).
These proteins have also been shown to mediate actin-independent signaling cascades. These include (i) gene expression by activation of the serum response factor (SRF) which along with ternary complex factors (TCFs) interact with serum response elements found in certain gene promoters like c-fos, (ii) cell cycle progression through G.sub.1 phase and (iii) induction of tumorigenic transformation of NIH 3T3 and Rat1 rodent fibroblasts (Khosravi-Far et al., Adv. Cancer Res., 1998, 72, 57-107).
The elevated expression of RhoC has been demonstrated in a megakaryocytic leukemia cell line, CMK. CMK cells undergo a process of polyploidization called endomitosis which results in platelet production. These cells were shown to express high levels of RhoC thereby linking RhoC to the regulation of megakaryocyte function(Takada et al., Exp. Hematol., 1996, 24, 524-530).
Manifestations of altered RhoC regulation appear in both injury and disease states with a role indicated for RhoC in the development of cancer. Cellular transformation and acquisition of the metastatic phenotype are the two main changes normal cells undergo during the progression to cancer. In recent studies of ductal adenocarcinoma of the pancreas, RhoC was shown to be expressed at higher concentrations in tumor cells than in non-tumor cells. In addition, metastatic lesions and tumors that showed lymph node metastasis also exhibited elevated levels of RhoC. Taken together, these results suggest that the overexpression of RhoC is involved in pancreatic carcinogenesis (Suwa et al., Br. J. Cancer, 1998, 77, 147-152).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of RhoC. To date, strategies aimed at inhibiting RhoC function have involved the use of bacterial enzymes such as the Clostridium botulinum C3 exoenzyme which ADP ribosylates the protein rendering it inactive or agents (natural enzyme inhibitors) to inhibit the posttranslational modification (isoprenylation) of RhoC (Narumiya and Morii, Cell Signal, 1993, 5, 9-19). However, these targeting strategies are not specific to RhoC, as many proteins undergo similar posttranslational modifications. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting RhoC function.